1. Field of the Invention
The present disclosure pertains to methods for identification of plant species using molecular genetic tools. More particularly, the disclosure relates to validation of Echinacea species through DNA polymerase chain reaction (PCR) using primers designed based on their information content.
2. Description of Related Art
The use of dietary supplements, particularly vitamins and botanicals, have become very popular in the United States and Europe. A recent survey conducted by the FDA found that 16 million Americans use botanical supplements. The industry has consistently grown at a rate of approximately 25% annually since 1990. Supplements containing the Purple Coneflower, Echinacea purpurea and other related species, is one of the top selling products. A survey published in USA Today indicated that 19% of adult Americans have used Echinacea for the treatment of colds and flu symptoms. The plant contains substances that have been shown to non-specifically stimulate the immune system in animals and man (Stimpel et al. 1984; Roesler et al. 1991a, 199b). Bioactive high-molecular-weight polysaccharides are believed to be involved in a stimulation of lymphocytic macrophages and B cells with the production of various cytokines (e.g. interleukins, tumor necrosis factor and interferons). The response has been measured in-vivo and verified in clinical studies (Braunig et al 1992; Schoneberger et al. 1992). These plant used in its typical dosage forms has also been shown to non-toxic with no measurable side effects or known contraindications (Mengs et al. 1991).
The North American genus Echinacea consists of nine species and multiple varieties and has a distribution centering on Arkansas, Oklahoma, Missouri and Kansas. While the species are relatively distinct from one another, specific differences are narrowly defined. All taxa will hybridize when brought together and considerable natural hybridization occurs (McGregor 1968). E. angustifolia and E. atrorubens, which show geographic introgression, exhibit considerable overlap in stem and petiole structures. Hybrids of E. simulata and E. sanguinea appear to be very similar to the Arkansas race of E. pallida, and E. simulata has in the past been considered a variety of E. pallida. Further studies revealed differences in pollen size and morphology and that pallida was polyploid, and those natural hybrids were triploid and sterile. Because of hybridization, similarities between species and overlapping ranges, the identification of Echinacea species based on morphological characteristics alone is a complex process that is prone to ambiguity.
The currently practiced methods for identification of botanical species from processed plant materials (dried roots, stems, leaves, flower and seed) involve the chemical identification of chromatographic profiles of plant secondary products. These chromatographic techniques include thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC). The identification of a plant species is accomplished by comparison of chemical constituent profiles with chemical standard mixtures derived from extracts of a voucher plant specimen. Confirmations are often made by microscopic evaluation of the processed plant material. The results obtained from such analysis are subject error and misinterpretation because many plant secondary products are common within families and genera, and their natural variability can be influenced by growth cycle and habitat.
Public safety and product efficacy demand uniform state-of-the-art quality assurance programs. The Food & Drug Administration has a mandate to begin regulation of the dietary supplement industry for quality assurance and good manufacturing practices (US Code, 63 Fed. Reg. 23624). With the implementation of regulations to ensure quality control of product manufacture of phytomedicinals (21 CFR 201.128, Food & Drug Cosmetic Act), species certification is likely to be required of all botanical raw materials. Even if not required by the FDA, such certification increases consumer confidence and provides a competitive market advantage for producers of the certified product. It is also conceivable that, certification may become contractually mandated by international importers of phytomedicinals or by traders in commodity futures markets.
Molecular genetic analysis of highly conserved genes have been used in species identification because distinct genetic sequences are found in different species and in some instances individuals within the same species. These profiles can be determined with great precision and when properly applied are reproducible, rapid and scalable to large numbers of specimens. In addition to DNA sequence analysis, various DNA-based methods have been used to identify differences among individuals, populations and plant or animal species. In plant taxonomy, the techniques of RFLP (Restriction Fragment Length Polymorphism) and AFLP (Amplified Fragment Length Polymorphisms) have been used to characterize genetic differences between species and their sub-populations. Both procedures generate an array of DNA fragments of varying lengths is generated by these procedures that are unique for a given individual organism. The RFLP method is does not provide optimal discrimination of species (Hollis et al. 1999), requires highly purified DNA, and is not easily scaled to large numbers of specimens and can give erroneous results due to epigenetic differences between species (Jupe and Zimmer 1990). AFLP has been used to characterize the taxonomy of the genus of Echinacea (Baum et al. 2002). This technique, however, can be difficult to reproduce in different laboratories. This may be due to the fact that the AFLPs are derived from highly repetitive sequences that are not stable in plant genomes (Reamon-Buttner S M et al. 1999).